Fluorometer

ABSTRACT

In order to increase measuring sensitivity of a fluorometer, a transmitter of monochromatic test light beam is arranged opposite a receiver of a measuring light beam. A measuring chamber including a fluorescent indicator space is arranged between the transmitter and the receiver. The distance between the transmitter and the receiver is adjustable to match to the thickness of the measuring chamber.

BACKGROUND OF THE INVENTION

The present invention relates in general to a fluorometer and inparticular to a fluorometer of the type including a transmitter of amonochromatic light beam, a receiver of a monochromatic light beam, anda measuring chamber enclosing a space containing fluorescent indicatorsand including means for introducing an object of measurement into aneffective contact with the indicator space.

The measuring principle of the fluorometer of this kind is based onquenching fluorescent radiation due to the effect of physical orchemical parameters of articles under measurement on the fluorescentradiation of the indicator. In this manner, changes in concentration orof parameters of the object of measurement can be determined. It is thequenching constant K defining the measuring sensitivity of theindicators for the concentration or parameter changes of the measuredsubstance which is characteristic for the measuring process of thiskind.

Fluorometers having the above-described features are extensively used inthose fields where diverse measuring tasks are to be accomplished withthe same measuring device. For different measuring requirements, it issufficient to exchange the measuring chamber or the indicator space inthe chamber only while the optical and light measuring devices remainthe same.

Nevertheless, there are measuring tasks such as for example consecutivemeasurements in medical and biological fields in which highlyspecialized measuring devices are needed having a technological qualitywhich is best suited to these measuring problems.

Especially in measuring transparent media, both solid or liquid orgaseous which exhibit only a minute fluorescent radiation, atransluminescent measuring device is employed with advantage.Fluoroscopy or measurement by transillumination is known from U.S. Pat.Nos. 3,725,658, and 3,612,866.

SUMMARY OF THE INVENTION

A general object of this invention is to improve the measurement usingfluoroscopy.

In particular, it is an object of the invention to provide an improvedfluorometer in which a maximum solid angle of the fluorescent radiationis effectively utilized.

Another object of this invention is to improve signal to noise ratio ofthe output signal.

In keeping with these objects and others which will become apparenthereafter, one feature of the invention resides, in the provision of afluorometer which comprises a transmitter of a monochromatic light beam,a receiver of a monochromatic light beam, a measuring chamber includingtwo opposite windows delimiting an indication space therebetween, theindicator space containing fluorescent indicators, and means forintroducing an object of measurement in effective contact with theindicator space, and the transmitter and receiver of a monochromaticlight beam being arranged adjacent respective windows so that thedistance between the transmitter and the receiver correspondsubstantially to the thickness of the measuring chamber.

By virtue of this arrangement, the receiver is exposed to radiation fromthe indicator space at a maximum solid angle which is limited only bythe thickness of the layer of the object of measurement in the measuringchamber.

The resulting substantial improvement of the signal to noise ratio ofthe output signal permits the use of indicators which hitherto due totheir inferior measuring sensitivity could not be employed in manyapplications. Since the invention makes it possible to select asubstantially increased number of indicators, it is now possible tomeasure types of particles or physical or chemical parameters whichcannot be employed in prior art fluorometers.

For example, the novel measuring arrangement is operable withfluorescent indicators having quenching constant (K value) of 10⁻³ /Torrto 10⁻⁵ /Torr, whereas prior art measuring arrangements have beenlimited to indicators having K value between 10⁻¹ /Torr to 10⁻³ /Torr inorder to generate detectable signals.

Moreover, it is possible to use conventional sensitive fluorescenceindicators in combination with transmitters emitting a light beam at alonger wavelength to which the indicators are less sensitive. Thiscombination opens the possibility to make use of long wave transmitters,for example, light emitting diodes and also semiconductive lightreceiver. Due to the relatively limited intensity or photosensitivity oflight emitting diodes, photodiodes or phototransmittors, the latterdevices hitherto did not find application for operation in long waveranges.

The monochromatization can be accomplished without problems by means ofa filter. It is true that a filter diminishes the light signalintensity, nevertheless this drawback is freely compensated by thesubstantially increased sensitivity due to the arrangement of thisinvention. In the case when it is possible to employ the same wavelength ranges both at the transmitter and at the receiver, the filterscan be dispensed with.

While employing semiconductive elements for the light transmitters andreceivers, it is now possible to design measuring devices which aresubstantially reduced in size, have a compact construction and a smallconsumption of energy. Consequently, portable measuring devices can beeasily designed.

Since this invention enables the use of less sensitive fluoresentindicators, long time stability of the measuring arrangement issubstantially higher than in prior art devices using sensitiveindicators. Similarly, the linearity of less sensitive indicators issuperior than that of the sensitive indicators. The latter features ofthe device of this invention represent substantial advantages inmeasuring technology.

Still another advantage is the increase of the measuring range during pHmeasurements. In conventional arrangements of this kind only narrow pHranges, namely around the pH value of about 1.5 pH units are measurablebecause the intensity outside this narrow range considerably decreases.Since this invention enables the use of high radiation intensities, thislimitation is no longer an obstacle and it is possible to measure alsoin pH ranges which extend far beyond the above pH values. Moreover, incomparison with prior art devices, this invention enables themeasurement of thinner layers of the measured substance, thus achievingan improvement in the response time.

In further elaboration of this invention, the distance between the lighttransmitter and the measured light receiver is adjustable. In thismanner, a minimum distance between the transmitter and the receiver ismaintained even when using exchangeable measuring chambers of differentthickness.

In addition, the thickness of the measuring chamber itself isadjustable. This modification results in a further optimization of thesignal intensity.

With advantage, the indicator space adjoins the window of the measuringchamber which faces the light receiver so that the solid angle ofreceived radiation is maximal. The concomittant decrease of the solidangle at the side of the transmitter is compensated by increasedintensity of transmitted radiation.

Still another improvement of the measuring arrangement of this inventionis achieved by designing the indicator space as a locally variable Kvalue (quenching constant value) and shiftably arranging this indicatorspace within the measuring chamber.

By shifting such a variator of the quenching constant K relative to thepath of the transmitted light beam, the quenching constant K isadjustable and can be optimized for a particular measurement.

The novel features which are considered as characteristic for theinvention are set forth in particular in the appended claims. Theinvention itself, however, both as to its construction and its method ofoperation, together with additional objects and advantages thereof, willbe best understood from the following description of specificembodiments when read in connection with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a measuring arrangement according to this invention;

FIG. 2 is a modification including a reference chamber;

FIG. 3 is a plot diagram showing radiation intensity of a light emittingdiode versus wavelength;

FIG. 4 illustrates an arrangement of this invention employing a variatorof quenching constant K; and

FIG. 4a is a plot diagram illustrating the relationship of K values todifferent lengths of the indicator space.

DESCRIPTION OF THE PREFERED EMBODIMENTS

In the arrangement of the fluorometer illustrated in FIG. 1, a lightbeam transmitter in the form a light emitting diode 10 is provided onits output side 11 with a monochromatic filter 11 to emit a beam ofmonochromatic light toward a juxtaposed photoelectric receiver 20. Theoutput signal A from the receiver is indicated on an electric indicator.Source of electric power B power supplies the entire measuringarrangement. The input side of the photoelectric receiver 20 is alsoprovided with a monochromatic filter 21, forming together amonochromatic receiver 2. The monochromatic light beam transmitterconstituted by the light emitting diode and the monochromatic filter 11is indicated by reference numeral 1. A measuring chamber 3, either inthe form of a vessel or of a throughflow chamber, is arranged betweenthe transmitter 1 and the receiver 2.

The illustrated measuring chamber 3 is designed with a relatively largecross-section so that a gas stream G which in this example representsand object of measurement, can be directed therethrough withoutobstructions. This kind of measuring chamber is suitable, for examplefor use in a breath analyzer which due to a small constant of thecompact fluorometer of this invention can be readily realized.

With suitable modifications, the fluorometer can be also used formeasuring transparent liquids.

To monitor the throughflow of the fluid in the measuring chamber, aflowmeter E can be used and a thermostat for stabilizing the temperatureis also of advantage.

Preferably, the monochromatic transmitter 1 and/or the monochromaticreceiver 2 are adjustably arranged on a base plate 4 so that both unitscan be adjusted as close to the measuring chamber 3 as possible. In amodification, the measuring chamber 3 and one of the units 1 or 2 areadjustably supported on the base plate.

As mentioned before, both the light emitting diode 10 and thephotoelectric receiver 20 are preferably in the form of semiconductivedevices which both electrically and mechanically have superiormechanical and electrical quality. The disadvantage of semiconductivedevices, namely the fact that they are most effective in the long waverange of the light spectrum, whereas most of the fluorescent indicatorsin the measuring chamber 3 operate in the short wave portion of thespectrum, as illustrated in FIG. 3, is compensated by high intensity oflight radiation of the arrangement of this invention.

In the measuring chamber 3 there is provided an indicator space 31frequently called an optode, which is in effective contact with thegaseous stream G. The optical properties of fluorescent indicatorsarranged in the indicator space 31 may vary in a known manner due to theinteraction with the gaseous stream G, and these changes in the excitedfluorescent light are detected by the monochromatic light receiver 2.

The intensity of the fluorescent radiation from the indicator space 31can be increased by making the side walls 3' of the measuring chamberelastic and adjusting the thickness of the measuring chamber 3 by meansof an adjustment mechanism 34 until a maximum output signal at theindicator I is obtained. In this manner, the measuring chamber isadjusted to its optimum thickness.

Windows 35 and 36 of the measuring chamber 3 facing respectively thetransmitter 1 and the receiver 2, can be made in the form of opticalmonochromatic filters replacing the filters 11 and 21. The fluorescentindicators arranged in the indicator space 31 preferably have such aconcentration that the excitation light beam from the transmitter isfully absorbed in the indicator space. In both cases the space betweenthe photoelectric transmitter 10 and photoelectric receiver 20 (whichneed not be monochromatic) is reduced and consequently the intensity ofmeasured light is increased. The mechanical adjustment device 34 whichin this example is in the form of adjusting screws, threadingly engaginga holder of the measuring chamber are illustrated on an enlarged scaleby way of an example only and in practice the holder is as thin aspossible or constructed such as not to interfere with the adjustment ofthe distance between the transmitting and receiving units.

Provided that the thickness of the measuring chamber must not fall belowa certain value, as is the case in a breath analyzer, then it is ofadvantage to focus the transmitted light beam by optical means 40 asillustrated in FIG. 4. The optical means are, for example, fresnelplates or lenses.

As can be seen from FIG. 3, the wavelength of radiation emitted by acold light transmitter 1, such as, for example, light emitting diode 10,overlaps with the wavelength of applicable fluorescent indicators inmeasuring chamber 3 over a very narrow band only. In particular theexcitation radiation for the indicators (curve 1), the fluorescentradiation of the indicators (curve 3), and the light emission of thelight emitting diode (curve 2) overlap at a relatively low intensity inthe wavelength range of about 470 nm. The low intensities in this caseare compensated for by the high signal gain of the arrangement of thisinvention.

If desired, it is possible to use also different radiation sources whenthe resulting heat is withdrawn by suitable conventional means.

In the embodiment illustrated in FIG. 2, there is provided parallel tothe path of flow of the object of measurement, a reference chamber 60communicating with the measuring chamber 30. This arrangement enablesthe generator of a measuring signal M which is independent of theabsolute fluctuations of radiation intensity. The measuring signal M iscomputed in a conventional manner, for example by means of quotientformation in a computer C of a reference signal R derived from thereference chamber 60, and the measuring signal A' derived in themeasuring chamber 30. The reference chamber 60 can operate either understandard conditions independently of the object of measurement or as inthe illustrated example, the measured substance flows also through thereference chamber. In the latter case, the indicator space in thereference chamber must be designed such as to remain unaffected by themeasured substance.

In another modification, shown in FIGS. 4 and 4a, the indicator space 33has locally differentiated K values (quenching values) as shown on theabscissa in the plot diagram of FIG. 4a. In the diagram, the verticalaxis 33" corresponds to the length of the indicator space 33 and a scaleof K values starting from an initial value K_(o), is on the horizontalaxis K'. The K values at different locations of the indicator space areindicated by plotted line K". In this case the indicator space isshiftable relative to the light beam 37 in the direction of arrow 38 andrepresents the beforementioned variator of the quenching constant K. Theposition adjustment of the indicator space is made in response to thenoise component of the output signal A" applied to a indicator. Thecorrect K value is adjusted when the noise signal component isminimized.

For clarification of the K value adjustment it will be noted that thefluorescent quenching, according to Stern-Vollmer proceeds according tothe following equation:

    I(p)=I.sub.o /1+Kp                                         (1)

wherein I_(o) is fluorescence intensity in the absence of the object ofmeasurement, I(p) is fluorescence in the presence of the object ofmeasurement, K is quenching constant and p is concentration value of theobject of measurement.

From the above equation, the measuring sensitivity S(P) results as:

    S(P)=dI/dp=k·I.sub.o (1+Kp).sup.2.                (b 2)

The functions S(P) representing the measuring sensitivity for variousvalues of the quenching constant K intersect in the range of largermeasuring values. In this manner, by adjusting the K values (for exampleby locally differentiated material composition of the indicator space),the noise component of the output signal can be minimized for aparticular measuring value.

From the above equation (1) a linear form of the measuring sensitivityor fluorescent indicators of low sensivity results as follows:

    I(p)=(1-K·p)I.sub.o.                              (3)

It will be understood that each of the elements described above, or twoor more together, may also find a useful application in other types ofconstructions differing from the types described above.

While the invention has been illustrated and described as embodied inspecific examples of fluorometric arrangement, it is not intended to belimited to the details shown, since various modifications and structuralchanges may be made without departing in any way from the spirit of thepresent invention.

Without further analysis, the foregoing will so fully reveal the gist ofthe present invention that others can, by applying current knowledge,readily adapt it for various applications without omitting featuresthat, from the standpoint of prior art, fairly constitute essentialcharacteristics of the generic or specific aspects of this invention.

What is claimed as new and desired to be protected by Letters Patent isset forth in the appended claims:
 1. A fluorometer comprising atransmitter of a monochromatic light beam, the transmitter including asemi-conductive light emitting diode, a receiver of a monochromaticlight beam, the receiver including a semiconductive photoelectricdevice, a measuring chamber enclosing an indicator space includingfluorescent indicating means, and means for introducing an object ofmeasurement in effective contact with the indicator space, thetransmitter having a light output side and the receiver having a lightinput side, said light output and input sides being arranged oppositeeach other and the measuring chamber being arranged therebetween so thatthe distance between the transmitter and the receiver correspondssubstantially to the thickness of the measuring chamber, and means foradjusting spacing between said transmitter, said measuring chamber andsaid receiver.
 2. A fluorometer as defined in claim 1, wherein both thelight emitting diode and the semiconductive photoelectric device includea monochromatic filter.
 3. A fluorometer as defined in claim 1, whereinthe fluorescent indicating means includes fluorescent indicators whosequenching coefficient is locally differentiated, and the indicator spacebeing shiftable transversely with respect to the light beam from thetransmitter.
 4. A fluorometer as defined in claim 1, wherein thefluorescent indicating means includes fluorescent indicators whosequenching constant is less than 10⁻³ Torr.
 5. A fluorometer as definedin claim 1, wherein the measuring chamber communicates with saidintroducing means is in the form of a throughflow chamber.
 6. Afluorometer as defined in claim 5 wherein said introducing meansincludes a flowmeter.
 7. A fluorometer as defined in claim 1, whereinthe measuring chamber includes opposite windows facing respectively thetransmitter and the receiver, the windows being in the form ofmonochromatic filters.
 8. A fluorometer as defined in claim 1, whereinthe measuring chamber communicates with a reference chamber includingfluorescent indicating means defining a reference indicator space, areference monochromatic light receiver arranged opposite the referenceindicator space, the monochromatic light transmitter being positionedand arranged to transmit a test light beam both to the measuring chamberand to the reference chamber, and means for computing a quotient fromoutput signals of the measuring light receiver and the reference lightreceiver to provide an indication which is independent of fluctuationsof the light intensity.
 9. A fluorometer as defined in claim 8, whereinthe fluorescent indicating means includes fluorescent indicators at aconcentration which absorbs the monochromatic test light beam before itreaches the light receiver.
 10. A fluorometer as defined in claim 8,further comprising means for focussing the test light beam from thetransmitter.
 11. A fluorometer as defined in claim 1 further comprisingmeans for adjusting the thickness of said measuring chamber.
 12. Afluorometer as defined in claim 1 wherein said indicator space isarranged at a side of said measuring chamber facing said receiver.